JP2023513800A - HUMAN 4-1BB AGONIST ANTIBODY AND METHODS OF USE THEREOF - Google Patents

Abstract

An isolated or recombinant monoclonal antibody that binds to 4-1BB is provided. In some cases, the antibodies of embodiments can be used for the detection, diagnosis and/or therapeutic treatment of human diseases such as cancer. A growing body of evidence indicates that anti-4-1BB monoclonal antibodies have strong anti-tumor properties, which themselves are due to their potent CD8+ T-cell activation, IFN-γ production, and ability to induce cytolytic markers. is the result of However, there is an unmet need for improved human 4-1BB antibodies. The present disclosure provides a means of solving this problem.

Description

This application claims the benefit of US Provisional Patent Application No. 62/977,658, filed February 17, 2020, which is hereby incorporated by reference in its entirety.

INCORPORATION OF THE SEQUENCE LISTING The Sequence Listing, 23 KB (measured on Microsoft Windows®) created on February 16, 2021, contained in a file named "UTFCP1467WO_ST25.txt", is hereby incorporated by electronic transmission. and incorporated herein by reference.
Background 1. FIELD The present invention relates generally to the field of molecular biology. More specifically, it relates to tumor necrosis factor superfamily receptor (TNF-FSFR; 4-1BB; CD137) agonist antibodies.

2. Description of the Related Art 4-1BB (CD137), a member of the TNF receptor superfamily, is an activation-inducing T cell co-stimulatory molecule (Vinay and Kwon, 2012). Signaling through 4-1BB upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the 4-1BB pathway has been emphasized in many diseases, including cancer. A growing body of evidence indicates that anti-4-1BB monoclonal antibodies have strong anti-tumor properties and, as such, are characterized by their potent CD8 ⁺ T cell activation, IFN-γ production, and cytolytic markers. This is the result of inducibility. Moreover, combination therapy with anti-4-1BB and other anti-cancer therapies such as radiation has robust tumor regression potential against non-immunogenic or poorly immunogenic tumors. Moreover, adoptive transfer of ex vivo anti-4-1BB-activated CD8 ⁺ T cells from previously tumor-treated animals efficiently promoted tumor progression in recipient mice inoculated with fresh tumors. impede. Furthermore, targeting of tumors by variants of 4-1BBL relative to 4-1BB also has potent anti-tumor effects. However, there is an unmet need for improved human 4-1BB antibodies.

Vinay and Kwon, Mol Cancer Ther, 11(5): 1062-70, 2012.

Overview In a first embodiment, the present disclosure provides an isolated monoclonal antibody that specifically binds to 4-1BB, wherein (a) the first V _H CDR is SEQ ID NO:3; (b) the second V _H CDR is identical to SEQ ID NO: 5; (c) the third V _H CDR is identical to SEQ ID NO: 7; (d) the first V _L CDR is is identical to SEQ ID NO: 11, (e) the second V _L CDR is identical to SEQ ID NO: 13, and (f) the third V _L CDR is identical to SEQ ID NO: 15, or (a) the first _VH CDR is identical to SEQ ID NO: 19, (b) the second _VH CDR is identical to SEQ ID NO:21, (c) the third _VH CDR is identical to SEQ ID NO:23 , (d) the first V _L CDR is identical to SEQ ID NO: 27, (e) the second V _L CDR is identical to SEQ ID NO: 29, and (f) the third V _L CDR is SEQ ID NO: 31, or (a) the first _VH CDR is identical to SEQ ID NO:35, (b) the second _VH CDR is identical to SEQ ID NO:37, (c) the third The V _H CDR is identical to SEQ ID NO: 39, (d) the first V _L CDR is identical to SEQ ID NO: 43, (e) the second V _L CDR is identical to SEQ ID NO: 45, and ( f) the third V _L CDR is identical to SEQ ID NO: 47, or (a) the first V _H CDR is identical to SEQ ID NO: 51 and (b) the second V _H CDR is SEQ ID NO: 53 (c) the third V _H CDR is identical to SEQ ID NO: 55; (d) the first V _L CDR is identical to SEQ ID NO: 59; (e) the second V _L CDR is is identical to SEQ ID NO:61; and (f) the third V _L CDR is identical to SEQ ID NO:63.

In some aspects, the antibody is at least about 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, to the _VH domain of clone 54 (SEQ ID NO:2) %, 97%, 98%, 99% or 100% identical _VH domain and at least about 80% identical to the VL domain of clone 54 ( _SEQ ID NO: 10), such as 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical _VL domains. In a particular aspect, the antibody comprises a _VH domain identical to the VH domain of clone 54 (SEQ ID _NO :2) and a _VL domain identical to the _VL domain of clone 54 (SEQ ID NO:10).

In particular aspects, the antibody is at least about 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, to the _VH domain of clone 135B (SEQ ID NO: 18) , a V _H domain that is 97%, 98%, 99% or 100% identical, and a V _L domain that is at least about 80% identical, such as 85%, 90%, 91%, 92%, to the VL domain of clone 135B (SEQ ID NO:26); 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical _VL domains. In a particular aspect, the antibody comprises a _VH domain identical to the _VH domain of clone 135B (SEQ ID NO:18) and a _VL domain identical to the _VL domain of clone 135B (SEQ ID NO:26).

In some aspects, the antibody is at least about 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, to the _VH domain of clone 138 (SEQ ID NO:34) %, 97%, 98%, 99% or 100% identical _VH domain and at least about 80% identical to the _VL domain of clone 138 (SEQ ID NO:42), e.g., 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical _VL domains. In a particular aspect, the antibody comprises a _VH domain identical to the VH domain _of clone 138 (SEQ ID NO:34) and a _VL domain identical to the _VL domain of clone 138 (SEQ ID NO:42).

In some aspects, the antibody is at least about 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, to the _VH domain of clone 49A (SEQ ID NO:50) %, 97%, 98%, 99% or 100% identical _VH domain and at least about 80% identical to the _VL domain of clone 49A (SEQ ID NO:58), such as 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical _VL domains. In a particular aspect, the antibody comprises a _VH domain identical to the VH domain _of clone 49A (SEQ ID NO:50) and a _VL domain identical to the _VL domain of clone 49A (SEQ ID NO:58).

In certain aspects, the antibody is recombinant. In some aspects, the antibody is IgG, IgM, IgA or an antigen-binding fragment thereof. In particular aspects, the antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or single domain antibody. In certain aspects, the antibody is a human, humanized or deimmunized antibody. In a further aspect, the antibody is conjugated to an imaging agent, chemotherapeutic agent, toxin or radionuclide.

A further embodiment provides a composition comprising the antibody of this embodiment in a pharmaceutically acceptable carrier. Also provided herein is an isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of the embodiments.

Another embodiment is CDR1-3 of the _VH domain of clone 54 (SEQ ID NOS:3, 5 and 7), CDR1-3 of the _VH domain of clone 135B (SEQ ID NOS:19, 21 and 23), V of clone 138 A recombinant polypeptide is provided comprising an antibody _VH domain comprising the _H domain CDRs 1-3 (SEQ ID NOs: 35, 37 and 39) or the _VH domain CDRs 1-3 of clone 49A (SEQ ID NOs: 51, 53 and 55) do.

Further embodiments include CDRs 1-3 of 54 _VL domains (SEQ ID NOs: 11, 13 and 15), CDRs 1-3 of 135B _VL domains (SEQ ID NOs: 27, 29 and 31), 138 _VL domains Recombinant polypeptides are provided comprising an antibody V _L domain comprising CDRs 1-3 (SEQ ID NOS: 43, 45 and 47), or CDRs 1-3 of the V _L domain of 49A (SEQ ID NOS: 59, 61 and 63).

In a still further embodiment, the antibody _VH domain comprises CDR1-3 of the VH domain of clone 54 (SEQ ID NOS:3, 5 and 7) and CDR1-3 of the _VL domain of 54 (SEQ ID NOS:11, 13 and 15 ₎ . ); antibody V _H domain comprising CDRs 1-3 of the V _H domain of clone 135B (SEQ ID NOs: 19, 21 and 23) and CDRs 1-3 of the V _L domain of 135B (SEQ ID NOs: 27 _, 29 and 31); antibody V _H domains comprising CDRs 1-3 of the V _H domain of clone 138 (SEQ ID NOs: 35, 37 and 39) and CDRs 1-3 of the V _L domain of 138 (SEQ ID NOs: 43, 45 ₎ ; and 47); or an antibody V _H domain comprising the V _H domain CDRs 1-3 of clone 49A (SEQ ID NOS: 51, 53 and 55) and the V _L domain CDRs 1-3 of 49A (SEQ ID _NO : 59). , 61 and ₆₃ ) are provided.

A further embodiment provides an isolated polynucleotide molecule comprising a nucleic acid sequence encoding a polypeptide of the present embodiments. Host cells containing one or more polynucleotide molecules encoding the antibodies of the embodiments or recombinant polypeptides of the embodiments are also provided herein. In some aspects, the host cell is a mammalian cell, yeast cell, bacterial cell, pili cell, or insect cell.

Another embodiment is a method of producing an antibody comprising the steps of: (a) expressing in a cell one or more polynucleotide molecules encoding the V _L and V _H chains of the antibody of this embodiment; (b) purifying the antibody from the cells.

A further embodiment provides a method of treating a subject with cancer comprising administering to the subject an effective amount of an antibody of the present embodiments.

In some aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

In certain aspects, the antibody is in a pharmaceutically acceptable composition. In some aspects, the antibody is administered systemically. In some embodiments, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In a further aspect, the method further comprises administering at least a second anti-cancer therapy to the subject. In some aspects, the second anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. In certain aspects, the second anti-cancer therapy comprises adoptive T-cell therapy. In some aspects, the second anti-cancer therapy comprises immunotherapy. In certain aspects, the immunotherapy is an immune checkpoint inhibitor, such as anti-CTLA-4, anti-PD-L1 and/or anti-PD1 antibodies. In particular aspects, the immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) binding antagonist, a PDL1 binding antagonist or a PDL2 binding antagonist. In some aspects, the PD-1 binding antagonist is a monoclonal antibody or antigen-binding fragment thereof. In particular aspects, the PD-1 binding antagonist is nivolumab, pembrolizumab, CT-011, BMS 936559, MPDL328OA or AMP-224.

Other objects, features and advantages of the present invention will become apparent from the detailed description below. The detailed description and specific examples, however, while indicating specific embodiments of the invention, are merely intended so that various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It should be understood that it is given as an example.

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

Figures 1A-1B: Multiple high affinity anti-human 4-1BB antibodies were generated. (FIG. 1A) ELISA results of initial post-fusion antibody supernatant against recombinant human 4-1BB. (FIG. 1B) Titration curve of 4-1BB monoclonal antibody clone. Figures 1A-1B: Multiple high affinity anti-human 4-1BB antibodies were generated. (FIG. 1A) ELISA results of initial post-fusion antibody supernatant against recombinant human 4-1BB. (FIG. 1B) Titration curve of 4-1BB monoclonal antibody clone. Figures 1A-1B: Multiple high affinity anti-human 4-1BB antibodies were generated. (FIG. 1A) ELISA results of initial post-fusion antibody supernatant against recombinant human 4-1BB. (FIG. 1B) Titration curve of 4-1BB monoclonal antibody clone.

Figures 2A-2B: The present clone 54 4-1BB antibody (herein referred to as Curranlumab) amplifies human TILs with high efficiency. Two variants of clone 54 (Curranlumab) were used to amplify human tumor-infiltrating lymphocytes (TILs) from patient tumor masses, and the efficiency of each in generating a pure expanded CD8 ⁺ T cell product was evaluated using urelumab (BMS- 663513). Figures 2A-2B: The present clone 54 4-1BB antibody (herein referred to as Curranlumab) amplifies human TILs with high efficiency. Two variants of clone 54 (Curranlumab) were used to amplify human tumor-infiltrating lymphocytes (TILs) from patient tumor masses, and the efficiency of each in generating a pure expanded CD8 ⁺ T cell product was evaluated using urelumab (BMS- 663513).

Figure 3: PCR using several combinations of Ig variable domain primers. Asterisks indicate correct antibody transcripts for VH and VL chains. The remaining PCR bands observed are aberrant transcripts confirmed by sequencing.

Figures 4A-4B: Glioblastoma and colorectal cancer tumors were isolated and tumor-infiltrating T cells were amplified using the 41BB agonistic antibody clone 54 as either human IgG1 or mouse IgG2a. The extent of amplification, percent CD8 and percent CD4 are shown. Total TILs from glioblastoma and colorectal cancer patient tumors were amplified with clone 54 IgG1 and clone 54 IgG1 (FIG. 4A). Percentage of CD3+CD8+ TILs or CD3+CD4+ TILs generated by the proliferation method (Fig. 4B). Figures 4A-4B: Glioblastoma and colorectal cancer tumors were isolated and tumor-infiltrating T cells were amplified using the 41BB agonistic antibody clone 54 as either human IgG1 or mouse IgG2a. The extent of amplification, percent CD8 and percent CD4 are shown. Total TILs from glioblastoma and colorectal cancer patient tumors were amplified with clone 54 IgG1 and clone 54 IgG1 (FIG. 4A). Percentage of CD3+CD8+ TILs or CD3+CD4+ TILs generated by the proliferation method (Fig. 4B).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The tumor necrosis factor superfamily receptor (TNFSFR), also known as 4-1BB and CD137, costimulates the activation of myeloid cells, including T cells, NK cells, and antigen-presenting dendritic cells. do. For CD8 T cells, 4-1BB activation increases their proliferation, cytotoxicity, cytokine production, mitochondrial mass and viability. Therefore, in this study, 4-1BB agonistic antibodies were generated. To generate antibodies targeting human 4-1BB, mice were alternately immunized with cell and recombinant protein-based vaccines. Among the many lead candidates, the clone 54 4-1BB antibody that was sequenced and expressed in various mouse and human isotypes was selected. The present study found that the IgG2a version of clone 54 4-1BB antibody expanded and matured tumor-infiltrating CD8 T cells very efficiently.

Accordingly, in certain embodiments, the present disclosure provides human 4-1BB agonist antibodies, particularly 4-1BB IgG2a agonist monoclonal antibodies. The antibody may be manufactured under GMP-compliant conditions. The antibodies can be used in patients in monospecific or bispecific formats. In a further aspect, the antibody can be administered to cancer patients as immunotherapy alone or in combination with additional immunotherapies such as immune checkpoint inhibitors (eg, anti-PD1 or anti-CTLA4 antibodies).

I. DEFINITIONS As used herein, “essentially free” with respect to a specified ingredient is used herein to mean that none of the specified ingredients are intentionally included in the composition and/or Used as a contaminant or to mean present only in trace amounts. Therefore, the total amount of specific ingredients resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the amount of a particular ingredient cannot be detected by standard analytical methods.

As used herein, "a" or "an" may mean one or more. As used in the claims, the word "a" or "an" when used in conjunction with the word "comprising" can mean one or more.

The use of the term "or" in a claim is used to mean "and/or" unless explicitly indicated to refer only to the alternative or the alternatives are not mutually exclusive. However, this disclosure supports definitions that refer only to alternatives and to "and/or." As used herein, "another" may mean at least a second or a third or more. The term "about" generally means plus or minus 5% of the stated value.

"Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performing a procedure or modality on a subject for the purpose of obtaining therapeutic benefit for a disease or health-related condition.

"Subject" and "patient" refer to either humans or non-humans, such as primates, mammals and vertebrates. In certain embodiments, the subject is human.

The terms "therapeutic benefit" or "therapeutically effective" as used throughout this application refer to promoting or enhancing a subject's well-being with respect to medical treatment of this condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of disease. For example, treating cancer can include, for example, reducing the size of a tumor, reducing the aggressiveness of a tumor, slowing the rate of cancer growth, or preventing metastasis. Treating cancer can also refer to prolonging survival of a subject with cancer.

An "anti-cancer" agent is, for example, promoting cancer cell death, inducing cancer cell apoptosis, decreasing the rate of cancer cell proliferation, reducing the incidence or number of metastases, reducing tumor size inhibit tumor growth; reduce blood supply to tumors or cancer cells; promote immune responses to cancer cells or tumors; prevent or inhibit cancer progression; By prolonging the lifespan of a subject having it, the subject's cancer cells/tumors can be negatively affected.

The term "antibody" herein is used in the broadest sense, specifically monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , and antibody fragments so long as they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies that make up the population have the expected mutations, e.g. are identical except for naturally occurring mutations that may exist in Thus, the modifier "monoclonal" indicates that the antibody characteristic is not a mixture of individual antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies comprising target-binding polypeptide sequences, wherein the target-binding polypeptide sequence is a single target from a plurality of polypeptide sequences. Obtained by a process involving selection of binding polypeptide sequences. For example, the selection process can be the selection of unique clones from a plurality of clones such as pools of hybridoma clones, phage clones or recombinant DNA clones. The selected target binding sequence is, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo. It should be understood that further modifications may be made, such as to generate multispecific antibodies, to increase the sensitivity of the antibody, and antibodies comprising modified target binding sequences are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically not contaminated with other immunoglobulins.

The terms "contacted" and "exposed" refer to the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent, when applied to a cell, are delivered to or directly juxtaposed with the target cell. Used herein for description. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing.

The term "immune checkpoint" refers to a molecule, such as a protein, of the immune system that provides inhibitory signals to its components to balance the immune response. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. It is recognized in the art that pathways involving LAG3, BTLA, B7H3, B7H4, TIM3 and KIR constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (e.g. Pardoll , 2012; see Mellman et al., 2011).

"Immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes loss of function and complete blockade. In particular, the immune checkpoint protein is a human immune checkpoint protein. Immune checkpoint protein inhibitors are therefore in particular inhibitors of human immune checkpoint proteins.

II. Anti-41BB Antibodies In certain embodiments, antibodies or fragments thereof that bind to at least a portion of 4-1BB and activate signaling, eg, stimulate an immune response, are contemplated. As used herein, the term "antibody" refers to any immunological binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as antibody CDR domains that retain antigen-binding activity. is intended to refer broadly to polypeptides comprising Antibodies may be selected from the group consisting of chimeric antibodies, affinity matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments, or natural or synthetic ligands. Preferably, the anti-41BB antibody is a monoclonal or humanized antibody.

In some embodiments, the anti-41BB antibody comprises the heavy chain CDRs 1-3 of SEQ ID NO:2 (GYSFTDYN (SEQ ID NO:3), INPNYGTT (SEQ ID NO:5) and ARSPVEDYFDY (SEQ ID NO:7) and the light chain of SEQ ID NO:10). The anti-41BB antibody comprises SEQ ID NOs: 1, 2, 9 or 10 at least 80%, For example, they can have 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. SEQ ID NO: 17, 18, 25 or 26 and at least 80%, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% The anti-41BB antibody may have at least 80%, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, It may have 96%, 97%, 98%, 99% or 100% sequence identity The anti-41BB antibody has at least 80%, such as 85%, 90%, 91%, SEQ ID NO: 49, 50, 57 or 58 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity The heavy and light chain sequences are shown below.
Frame 1 (FR1) (Positions 77-505) Clone 54 4-1BB VH consensus sequence starting from IgG2a Nucleotide sequence:
GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTAACTATGGTACTACTAGCTACAATCAGAAGTTCAAGGGCAAGGCCACATTTACTGTAGACCAATCTTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCCGGTAGAGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTA（配列番号１）

Amino acid sequence:
EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATFTVDQSSSTAYMQLNSLTSEDSAVYYCARSPVEDYFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTL (SEQ ID NO: 2)

CDR1:
GYSFTDYN (SEQ ID NO: 3)
GGTTACTCATTCACTGACTACAAC (SEQ ID NO: 4)

CDR2:
INPNYGTT (SEQ ID NO: 5)
ATTAATCCTAACTATGGTACTACT (SEQ ID NO: 6)

CDR3:
ARSPVEDYFDY (SEQ ID NO: 7)
GCAAGATCCCCGGTAGAGGACTACTTTGACTAC (SEQ ID NO: 8)

VL consensus sequence nucleotide sequence starting from frame 1 (FR1) (positions 91-470):
GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGGTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTCATCACGTTCGGTGCTGGGACCAAGCTGGA
GCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT (SEQ ID NO: 9)

Amino acid sequence:
ENVLTQSPAIMSASPGEKVTMTCRARSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLITFGAGTKLELKRADAAPTVSIFPPSSEQ (SEQ ID NO: 10)

CDR1:
SSVSSSY (SEQ ID NO: 11)
TCAAGTGTAAGTTCCAGTTAC (SEQ ID NO: 12)

CDR2:
STS (SEQ ID NO: 13)
AGCACATCC (SEQ ID NO: 14)

CDR3:
QQYSGYPLIT (SEQ ID NO: 15)
CAGCAGTACAGTGGTTACCCACTCATCACG (SEQ ID NO: 16)

Frame 1 (FR1) (Positions 45-407) Clone 135B 4-1BB VH consensus sequence starting with IgG1 Nucleotide sequence:
AGGTGAAGCTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGATGGTACTACCTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG（配列番号１７）

Amino acid sequence:
VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNDGTTYYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSLYGSSYYFDYWGQGTTLTVSS (SEQ ID NO: 18)

CDR1:
GYTFTDYY (SEQ ID NO: 19)
GGATACACGTTCACTGACTACTAC (SEQ ID NO: 20)

CDR2:
INPNNDGT (SEQ ID NO: 21)
ATTAATCCTAACAATGATGGTACT (SEQ ID NO: 22)

CDR3:
ARSLYGSSYYFDY (SEQ ID NO: 23)
GCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTAC (SEQ ID NO: 24)

VL consensus sequence nucleotide sequence starting from frame 1 (FR1) (positions 46-379):
GATATTGTGATGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC（配列番号２５）

Amino acid sequence:
DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 26)

CDR1:
KSVSTSGYSY (SEQ ID NO: 27)
AAAAGTGTCAGTACATCTGGCTATAGTTAT (SEQ ID NO: 28)

CDR2:
LVS (SEQ ID NO:29)
CTTGTATCC (SEQ ID NO: 30)

CDR3:
QQSNEDPWT (SEQ ID NO: 31)
CAGCAAAGTAATGAGGACCCGTGGACG (SEQ ID NO: 32)

Frame 1 (FR1) (Positions 56-409) Clone 138 4-1BB VH consensus sequence starting with IgG2b Nucleotide sequence:
AGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAATTCCATCTACTATGCAGACACAGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCCTCGAATAATGGTTACTTCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG（配列番号３３）

Amino acid sequence:
VQLQESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLWVAYISSGSNSIYYADTVTGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCASNNGYFYFDYWGQGTTLTVSS (SEQ ID NO: 34)

CDR1:
GFTFSTYG (SEQ ID NO: 35)
GGATTCACTTTTCAGTGACTATTGGA (SEQ ID NO: 36)

CDR2:
ISSGSNSI (SEQ ID NO: 37)
ATTAGTAGTGGCAGTAATTCCATC (SEQ ID NO: 38)

CDR3:
ASNNGYFYFDY (SEQ ID NO: 39)
GCCTCGAATAATGGTTACTTCTACTTTGACTAC (SEQ ID NO: 40)

VL consensus sequence nucleotide sequence starting from frame 1 (FR1) (positions 63-378):
ATTGTGATCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC（配列番号４１）

Amino acid sequence:
IVITQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTVSRVEAEDAATYYCQQWSSDPFTFGSGTKLEIK (SEQ ID NO: 42)

CDR1:
SSVSY (SEQ ID NO: 43)
TCAAGTGTAAGTTAC (SEQ ID NO: 44)

CDR2:
ATS (SEQ ID NO: 45)
GCCACATCC (SEQ ID NO: 46)

CDR3:
QQWSSDPFT (SEQ ID NO: 47)
CAGCAGTGGAGTAGTGACCCATTCACG (SEQ ID NO: 48)

Frame 1 (FR1) (Positions 65-427) Clone 49A4-1BB VH consensus sequence starting with IgG1 Nucleotide sequence:
AGGTGAAACTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGGAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTTCTACCTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGAGAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTACTGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAG（配列番号４９）

Amino acid sequence:
VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKESHGKSLEWIGDINPNNGGSTYYNQKFKGKATLTVEKSSSTAFMELRSLTSEDSAVYYCARSLYGSTYYFDYWGQGTPLTVSS (SEQ ID NO: 50)

CDR1:
GYTFTDYY (SEQ ID NO: 51)
GGATACACGTTCACTGACTACTAC (SEQ ID NO: 52)

CDR2:
INPNNGGS (SEQ ID NO: 53)
ATTAATCCTAACAATGGTGGTTCT (SEQ ID NO: 54)

CDR3:
ARSLYGSTYYFDY (SEQ ID NO: 55)
GCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTAC (SEQ ID NO: 56)

VL consensus sequence nucleotide sequence starting from frame 1 (FR1) (positions 55-388):
GATATTGTGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAGCCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGGAACCTATTACTGTCAGCAAAGTAATGACGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC（配列番号５７）

Amino acid sequence:
DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAGTYYCQQSNDDPWTFGGGTKLEIK (SEQ ID NO: 58)

CDR1:
QSVDYDGDSY (SEQ ID NO: 59)
CAAAGTGTTGATTATGATGGTGATAGTTAT (SEQ ID NO: 60)

CDR2:
AAS (SEQ ID NO: 61)
GCTGCATCC (SEQ ID NO: 62)

CDR3:
QQSNDDPWT (SEQ ID NO: 63)
CAGCAAAGTAATGACGATCCGTGGACG (SEQ ID NO: 64)

Thus, polyclonal or monoclonal antibodies specific for 4-1BB, one or more of its respective epitopes, or any of the foregoing conjugates, by known means and as described herein Antibodies, antibody fragments, and binding domains and CDRs (including engineered forms of any of the foregoing), whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds. can be made independently of

Examples of antibody fragments suitable for this embodiment include, but are not limited to: (i) a Fab fragment consisting of the V _L , V _H , C _L and C _H1 domains; (ii) the V _H domain. (iii) an "Fv" fragment consisting of the V _L and V _H domains of a single antibody; ₍ iv) a "dAb" fragment consisting of the V _H domains; (v) an isolated (vi) F(ab′)2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) the _VH and _VL domains form the binding domain in which the two domains associate (viii) bispecific single-chain Fv dimers (U.S. Pat. No. 5,091,513 and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (US Patent Application Publication No. 20050214860). Fv, scFv, or diabody molecules can be stabilized by the incorporation of disulfide bridges linking the _VH and _VL domains. Minibodies containing scFvs attached to the CH3 domain can also be generated (Hu et al., 1996).

Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe 'antibody-like binding peptidomimetics' (ABiPs), which act as pared-down antibodies and have longer serum half-lives as well as less cumbersome synthesis. It is a peptide with particular advantages of the method.

Animals may be inoculated with an antigen such as the 4-1BB extracellular domain (ECD) protein to produce antibodies specific for 4-1BB. Antigens are often linked or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-proteinaceous substance attached to an antigen that is used to elicit an immune response in an animal. Antibodies produced in animals in response to challenge include different non-identical molecules (polyclonal antibodies) made from different individual antibody producing B lymphocytes. A polyclonal antibody is a mixed population of antibody species, each of which can recognize a different epitope on the same antigen. Given the correct conditions for polyclonal antibody production in animals, most of the antibodies in the animal's serum will recognize collective epitopes on the antigenic compounds against which the animal is immunized. This specificity is further enhanced by affinity purification, which selects only those antibodies that recognize the antigen or epitope of interest.

A monoclonal antibody is a single species antibody in which all antibody molecules recognize the same epitope, since all antibody-producing cells are derived from a single B-lymphocyte cell line. Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used in generating monoclonal antibodies. In some embodiments, rabbit, sheep or frog cells are used to generate monoclonal antibodies. The use of rats is well known and can offer certain advantages. Mice (eg, BALB/c mice) are routinely used and generally give a high rate of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from a mouse pre-immunized with the 4-1BB antigen and an immortal myeloma cell (usually a mouse myeloma). This technology provides a way to propagate a single antibody-producing cell for an indefinite number of generations so that unlimited amounts of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity can be produced. do.

Plasma B cells (CD45 ⁺ CD5 ⁻ CD19 ⁺ ) can be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for 4-1BB binding cells. After enrichment for antibody-producing B cells, total RNA can be isolated and cDNA synthesized. The antibody variable region DNA sequences from both heavy and light chains can be amplified, assembled into phage display Fab expression vectors, and transformed into E. coli. 4-1BB-specific binding Fabs can be selected by multiple rounds of enrichment panning and sequenced. Selected 4-1BB binding hits were expressed as full-length IgG in rabbit and rabbit/human chimeric formats using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and analyzed by fast protein liquid chromatography. Purification can be done using protein G resin with a graphic (FPLC) separation unit.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising antigen-binding sequences derived from a non-human donor grafted onto heterologous non-human, human, or humanized sequences (e.g., framework and/or constant domain sequences). is. Methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact. Alternatively, "fully human" monoclonal antibodies are produced in mice transgenic for human immunoglobulin genes. Methods for converting the variable domains of monoclonal antibodies to a more human form have also been developed by recombinantly constructing antibody variable domains having both rodent, eg, mouse and human, amino acid sequences. In a "humanized" monoclonal antibody, only the hypervariable CDRs are derived from a murine monoclonal antibody, and the framework and constant regions are human amino acid sequences (see U.S. Pat. Nos. 5,091,513, 6,881,557). It is derived from Replacing amino acid sequences in antibodies characteristic of rodents with amino acid sequences found at the corresponding positions in human antibodies is believed to reduce the potential for adverse immune reactions during therapeutic use. A hybridoma or other cell that produces an antibody may also undergo genetic mutation or other changes that may or may not alter the binding specificity of the antibody with which the hybridoma is produced. be.

Methods for producing polyclonal antibodies in various animal species, as well as various types of monoclonal antibodies, including humanized, chimeric and fully human, are well known and highly predictable in the art. For example, the following US patents and patent applications allow description of such methods. U.S. Patent Application Nos. 2004/0126828 and 2002/0172677; and U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024 . This specification and all patents, patent application publications, and other publications cited therein are hereby incorporated by reference into this application.

Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibodies are sheep, mouse (eg, mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. Additionally, newer technologies allow the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology is capable of producing specific antibodies in the absence of animal immunization, as described in US Pat. No. 6,946,546, incorporated herein by reference. enable. These techniques are further described in Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

Antibodies to 4-1BB are fully expected to have the ability to neutralize or antagonize the effects of 4-1BB, regardless of animal species, monoclonal cell line, or other source of antibody. Certain animal species may be less preferred for generating therapeutic antibodies, as they may be more likely to provoke allergic reactions due to activation of the complement system by the "Fc" portion of the antibody. However, whole antibodies can be enzymatically digested into "Fc" (complement binding) fragments and into antibody fragments with the binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antigen-antibody fragment will elicit an unwanted immunological response, and thus Fc-free antibodies may be preferentially used for prophylactic or therapeutic treatments. As noted above, antibodies may also be chimeric to reduce or eliminate adverse immunological consequences resulting from administering antibodies produced in other species or having sequences from other species to animals. or may be constructed to be partially or fully human.

Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within the protein, with or without loss of other functions or properties, of one of the polypeptides. or designed to adjust properties beyond that. Substitutions may be conservative, ie, one amino acid is replaced with one of similar shape and charge. arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; glutamate to aspartate; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; Tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically different residue, eg, a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa.

Proteins may be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is also contemplated that bacteria containing such variants may be embodied in compositions and methods. Therefore, it is not necessary to isolate the protein.

It is contemplated that from about 0.001 mg to about 10 mg/ml of total polypeptide, peptide and/or protein is present in the composition. Accordingly, the concentration of protein in the composition may be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.000. 5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any derivable range therein). of which about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% may be antibodies that bind 4-1BB.

Antibodies, or preferably immunological portions of antibodies, can be chemically conjugated to or expressed as fusion proteins with other proteins. For the purposes of this specification and the appended claims, all such fusion proteins are included within the definition of an antibody or immunological portion of an antibody.

Embodiments provide antibodies and antibody-like molecules against 4-1BB, polypeptides and peptides linked to at least one agent to form an antibody conjugate or payload. In order to enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is conventional practice to link or covalently bind or conjugate at least one desired molecule or moiety. Such molecules or moieties can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules that have a desired activity, eg, cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands. , for example biotin.

Several methods are known in the art for attaching or conjugating antibodies to their conjugate moieties. Some attachment methods include, for example, diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycoluril-3 (tetrachloro -3-6?-diphenylglycouril-3), including the use of metal chelate complexes that employ organic chelating agents such as those attached to antibodies. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

II. Treatment of Diseases Certain aspects of the present embodiments can be used to prevent or treat diseases or disorders associated with 4-1BB signaling. The 4-1BB antibodies can be used to treat diseases such as cancer, infectious diseases, inflammatory diseases, or autoimmune diseases. Additional methods are provided for administering the vaccine. Suitable vaccines include, for example, tumor cell vaccines, DNA vaccines, GM-CSF modified tumor cell vaccines, or antigen loaded dendritic cell vaccines.

In certain embodiments, the compositions and methods of this embodiment comprise an antibody or antibody fragment against 4-1BB to activate its activity in cancer cell proliferation in combination with a second or additional therapy.

Accordingly, in some embodiments, provided herein are methods of treating cancer or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an anti-41BB antibody. Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, premalignant lesions of the lung. , colon cancer, melanoma and bladder cancer.

In some embodiments, the individual has a cancer that is resistant (demonstrated to be resistant) to one or more anti-cancer therapies. In some embodiments, resistance to anti-cancer therapy includes recurrence of cancer or refractory cancer. Recurrence can refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to anti-cancer therapy comprises cancer progression during treatment with anti-cancer therapy. In some embodiments, the cancer is in early stage or late stage.

A. Pharmaceutical Formulations When clinical applications of therapeutic compositions containing inhibitory antibodies are to be made, it will generally be beneficial to prepare a pharmaceutical or therapeutic composition appropriate for the intended application. In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of active compound. In other embodiments, the active compound may comprise from about 2% to about 75% by weight, or such as from about 25% to about 60%, and any derivable ranges therein.

The therapeutic compositions of this embodiment are advantageously administered in the form of injectable compositions, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. These preparations may be emulsified.

The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not provoke adverse, allergic, or other untoward reactions when administered to animals, including humans, as appropriate. . The preparation of pharmaceutical compositions containing the antibody or additional active ingredients will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (eg, human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, a "pharmaceutically acceptable carrier" includes any and all aqueous solvents such as water, alcoholic/aqueous solutions, saline, as known to those skilled in the art. , parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate, dispersion media, coatings, surfactants. , antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, Excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, such materials and combinations thereof are included. The pH and exact concentration of the various ingredients in the pharmaceutical composition are adjusted according to well-known parameters.

The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in subjects, each unit containing the desired dose as described above in connection with its administration, suitable route and treatment regimen. It contains a predetermined amount of therapeutic composition calculated to produce a response. The amount administered depends on the desired effect, both on the number of treatments and on the unit dose. The actual dosage of the compositions of the present embodiments administered to a patient or subject will depend on the body weight, age, health status and sex of the subject, type of disease being treated, extent of disease penetrance, prior or concurrent treatment physical and physiological factors such as therapeutic intervention, idiopathic disease of the patient, route of administration, and potency, stability and toxicity of the particular therapeutic agent. For example, doses may also range from about 1 μg/kg/body weight to about 1000 mg/kg/body weight per administration (such ranges include intervening doses) or more, and any range derivable therein. can contain. Non-limiting examples of ranges derivable from the numbers recited herein include ranges from about 5 μg/kg/body weight to about 100 mg/kg/body weight, from about 5 μg/kg/body weight to about 500 mg/kg/body weight. can be administered. The administering physician will, in any event, determine the concentration of active ingredient in the composition and appropriate dosage for the individual subject.

The active compounds can be formulated for parenteral administration, eg, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared either as liquid solutions or suspensions. Solid forms suitable for use in preparing solutions or suspensions with liquid prior to injection can also be prepared. The formulation can also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Proteinaceous compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, Formed with tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. can also be derived from

Pharmaceutical compositions can include solvents or dispersion media containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, maintenance of required particle size in the case of dispersions, and use of surfactants. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

B. Combination Treatment In certain embodiments, the compositions and methods of the present embodiments comprise an antibody or antibody fragment against 4-1BB to activate its activity in combination with a second or additional therapy. For example, the disease can be cancer.

Methods and compositions involving combination therapy enhance the therapeutic or protective effect and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to achieve a desired effect, such as killing cancer cells and/or inhibiting cell hyperproliferation. This process may involve contacting the cell with both the antibody or antibody fragment and a second therapeutic agent. The tissue, tumor or cells can be contacted with one or more compositions or pharmacological agents containing one or more agents (i.e., antibodies or antibody fragments or anti-cancer agents), or the tissue, tumor and/or or by contacting the cells with two or more different compositions or formulations, one composition being 1) an antibody or antibody fragment, 2) an anticancer agent, or 3) an antibody or antibody fragment. It provides both an anti-cancer drug and. It is also contemplated that such combination therapy can be used in combination with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

The terms "contacted" and "exposed" refer to the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent, when applied to a cell, are delivered to or directly juxtaposed with the target cell. Used herein for description. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing.

Inhibitory antibodies can be administered before, during, after anti-cancer treatment, or in various combinations with anti-cancer treatment. Administration can be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments in which the antibody or antibody fragment is provided to the patient separately from the anti-cancer agent, generally the time between each delivery is such that the two compounds are still able to exert an advantageous combined effect on the patient. is guaranteed not to pass a significant period of time. In such cases, it is contemplated that the antibody therapy and anti-cancer therapy can be provided to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 hours of each other. In some situations, days (2, 3, 4, 5, 6 or 7) to weeks (1, 2, 3, 4, 5, 6, 7 or 8) elapse between each administration. In some cases, it may be desirable to significantly extend the duration of treatment.

In certain embodiments, the course of treatment will last from 1 to 90 days or more (such ranges include intervening days). An agent may be administered on any day from day 1 to day 90 (such ranges include intervening days) or any combination thereof; It is contemplated to be administered on any day of the eye (such ranges include intervening days) or any combination thereof. Patients can receive single or multiple doses of the drug within a day (24 hours). Further, it is contemplated that after the course of treatment there will be a period of time during which no anti-cancer treatment is administered. This period may range from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months or more, depending on the patient's condition, such as prognosis, fitness, and health. (including intervening days) may continue. It is expected that treatment cycles will be repeated as necessary.

Various combinations can be used. In the example below, antibody therapy is "A" and anti-cancer therapy is "B."

Administration of any compound or therapeutic agent of the present embodiments to a patient follows general protocols for administration of such compounds, taking into account any toxicity of the drug. Thus, in some embodiments there is a step of monitoring toxicity resulting from combination therapy.

1. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to include a compound or composition administered in the treatment of cancer. These agents or drugs are categorized by their mode of action within cells, eg, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly cross-link DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimine and methylmelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine); acetogenins (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan) CC-1065 (including its adzelesin, carzelesin and vizelesin synthetic analogues); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogue, KW-2189); pancratistatin; sarcodictin; spongestatin; nitrogen mustards such as chlorambucil, chlornafadine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trophosfamide and uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, dynemycins (including dynemycin A); bisphosphonates such as clodronate; esperamycin; as well as the neocardinostatin chromophore and the related chromoprotein enediyne antibiotic chromophore, aclasino. mycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophylline, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin ( morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ethorubicin, idarubicin, marceromycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarmycin, olibomycin, peplomycin, potofilomycin (potfiromycin), puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamipurine and thioguanine; pyrimidine derivatives such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens, e.g. carsterone, dromostanolone propionate, epithiostanol, mepithiostane and testolactone; antiadrenergic e.g. mitotane and trilostane; folic acid supplements e.g. folinic acid; elformitin; elliptinium acetate; epothilone; etogluside; gallium nitrate; hydroxyurea; lentinan; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; lazoxan; rhizoxin; schizophyllan; spirogermanium; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobroman; gacytosine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; ibandronate; irinotecan (eg CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylhylornithine (DMFO); retinoids such as retinoic acid; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

2. Radiation Therapy Other agents that cause DNA damage and are widely used include what is commonly known as gamma rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors most likely affect a wide range of damage to DNA, its precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

3. Immunotherapy One skilled in the art will appreciate that additional immunotherapies can be used in combination or in conjunction with the method of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can include, for example, antibodies or chimeric antigen receptors (CARs) specific for some marker on the surface of tumor cells. In a further aspect, treatment may include administration of T cells or NK cells that target specific cancer cells. Such cells can be engineered or simply selected for anti-cancer cell activity.

In some embodiments, the antibody alone may act as an effector of therapy or may recruit other cells to actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) to serve as targeting agents. Alternatively, effectors can be lymphocytes bearing surface molecules that interact either directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. An antibody-drug conjugate (ADC) comprises a monoclonal antibody (MAb) covalently attached to a cell-killing drug. This approach combines the high specificity of MAbs for their antigen targets with highly potent cytotoxic drugs to create “armed” MAbs that deliver payloads (drugs) to tumor cells with concentrated levels of antigen. Bring. Targeted delivery of a drug also minimizes its exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The approval of two ADC drugs by the FDA, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, supports this approach. It was a demonstration. There are currently over 30 ADC drug candidates in various stages of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization become more and more mature, the discovery and development of new ADCs is increasingly dependent on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulation/high levels of expression and robust internalization in tumor cells.

In one aspect of immunotherapy, the tumor cells must possess some marker suitable for targeting, ie not present in the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. Immune stimulation including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT 3 ligand Molecules also exist.

Examples of immunotherapies currently under study or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. No. 5,801,005). and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies such as interferon alpha, beta and gamma, IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Pat. No. 5,830,880 and 5,846,945); monoclonal antibodies such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; US Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are molecules of the immune system that signal up (eg costimulatory molecules) or down signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2aR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic sexual T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3) and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

Immune checkpoint inhibitors can be drugs such as small molecules, recombinant forms of ligands or receptors, or, in particular, human antibodies (e.g., WO2015016718; Pardoll, 2012; both incorporated herein by reference). (incorporated herein). Known inhibitors of immune checkpoint proteins or analogues thereof may be used, particularly chimerized, humanized or humanized antibodies. As will be appreciated by those of skill in the art, alternative and/or equivalent designations may be used for the particular antibodies referred to in this disclosure. Such alternative and/or equivalent designations are interchangeable within the context of the present invention. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In particular aspects, the PD-1 ligand binding partner is PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In particular aspects, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. Antagonists can be antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins or oligopeptides. Exemplary antibodies are described in US Pat. No. 8,735,553, US Pat. No. 8,354,509 and US Pat. No. 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are in the art, such as those described in US Patent Application No. 20140294898, US Patent Application No. 2014022021 and US Patent Application No. 20110008369. , all of which are incorporated herein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, human, humanized, or chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (eg, an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (eg, the Fc region of an immunoglobulin sequence)). adhesin). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 drug described in WO 2006/121168. is an antibody. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.

Another immune checkpoint that can be targeted with the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence for human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein CD28 and both molecules bind CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen presenting cells. CTLA4 transmits inhibitory signals to T cells and CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for their function. T cell activation by the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., human, humanized, or chimeric antibody), antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. be.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in this method can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in US Pat. No. 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (also known as tremelimumab). CP675,206; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al., (2004) J Clin Oncology 22 (145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. 2001014424, 2000037504 and US Pat. No. 8017114, all incorporated herein by reference.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10 D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (eg, WO 1 /14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding and/or binds to the same epitope on CTLA-4 as the antibody above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with an antibody described above (eg, at least about 90%, 95% or 99% variable region identity with ipilimumab).

Other molecules for modulating CTLA-4 are described in US Pat. Nos. 5,844,905, 5,885,796 and International Patent Applications WO1995001994 and WO1998042752, all incorporated herein by reference. CTLA-4 ligands and receptors, such as, and immunoadhesion, such as those described in US Pat. No. 8,329,867, incorporated herein by reference.

4. Surgery Approximately 60% of people with cancer will undergo surgery of some type, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised and/or destroyed, and the treatments of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, It can be used in conjunction with other therapies such as immunotherapy and/or alternative therapies. Tumor resection refers to the physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

Removal of part or all of the cancerous cells, tissue, or tumor can form a cavity in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with additional anticancer therapy. Such treatment may be, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, It can be repeated every 8, 9, 10, 11 or 12 months. These treatments may also vary in dosage.

5. Other Agents It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, sensitizing hyperproliferative cells to apoptosis-inducing factors. Augmenting agents, or other biological agents are included. Increasing intercellular signaling by increasing the number of GAP junctions would increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents can be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative effects of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the susceptibility of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of this embodiment to improve treatment efficacy.

III. Kits and Diagnostics Various aspects of the embodiments contemplate kits containing therapeutic agents and/or other therapeutic agents and delivery agents. In some embodiments, this embodiment contemplates kits for preparing and/or administering the therapeutic agents of the embodiments. A kit may include one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments. Kits include, for example, at least one 4-1BB antibody and reagents for preparing, formulating and/or administering the components of the embodiments, or reagents for performing one or more steps of the methods according to the invention. can include In some embodiments, the kit may also include suitable containers, such as Eppendorf tubes, assay plates, syringes, bottles or tubes, which are non-reactive with the components of the kit. The container may be made from a sterilizable material such as plastic or glass.

The kit can further include instructions outlining the procedures of the methods described herein, following substantially the same procedures described herein or known to those skilled in the art. The instructional information may be in a computer readable medium containing machine readable instructions which, when executed using a computer, cause a display of an actual or hypothetical procedure for delivering a pharmaceutically effective amount of a therapeutic agent.

IV. Examples The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed in the examples which follow represent techniques found by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred forms for its practice. should be understood by those skilled in the art. However, one skilled in the art, in light of the present disclosure, can make many changes in the particular embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. It should be understood that

Example 1 - Production and Characterization of Anti-41BB Antibodies Mice were treated with human 4-1BB including clone 54 (IgG2a), clone 49A (IgG1), clone 138 (IgG2b), clone 151 (IgG3) and clone 111A (IgG1). (Fig. 1), cellular vaccines (macrophages or dendritic cells retrovirally engineered to express 4-1BB) and recombinant protein-based (recombinant with Fc or HIS tags) immunized alternately with the human 4-1BB extracellular domain) vaccine. Titration curves of clones were performed by ELISA and EC50 values were determined (Fig. 1B).

RNA extraction: Hybridoma cell pellets were provided by Long Vien (Monoclonal Antibody Core Facility MDACC). Total mRNA was extracted from hybridoma cell pellets. Total RNA was extracted from hybridoma clone 271-54 41-BB cell pellets using an RNA extraction protocol (Zymo Research).

RT-PCR: cDNA was generated from RNA by reverse transcription with random primers (RT Superscript III, Life Technologies). A PCR reaction that uses variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA, resulting in the bands in FIG. The VH and VL products were gel purified, cloned into the sequencing vector pCR™2.1-TOPO® and transformed into DH5a cells. Positive transformants were then selected using M16 forward and reverse primers (mFVIj forward primer (positions 59-76) sequence: ACTGCAGGTGTCCTCTCT (SEQ ID NO: 65); mRevIgG2a reverse primer (positions 526-506) sequence: TAACCCTTGACCAGGCATCC (SEQ ID NO: 66) mFVK4/5a forward primer (positions 47-67) sequence: TCAGCTTCYTGCTAATCAGTG (SEQ ID NO: 67); 1 mRK reverse primer (positions 491-471) sequence: ACTGAGGCACCTCCAGATGTT (SEQ ID NO: 68)). Colonies selected for miniprep plasmid purification were picked and analyzed by DNA sequencing (MDACC core facility).

Among the many lead candidates, the clone 54 4-1BB antibody that was sequenced and expressed in various mouse and human isotypes was selected. Patient tumor masses were cultured in tumor-infiltrating lymphocyte medium to support their survival and rapid growth in cell culture. This study demonstrates that the murine IgG2a version of the clone 54 4-1BB antibody expands and matures human tumor-infiltrating CD8 and CD4 T cells as efficiently as the anti-human 4-1BB antibody urelumab (Fig. 2B). Found it. Moreover, clone 54 efficiently amplified tumor-infiltrating CD8 and CD4 T cells from tumors of patients with glioblastoma or colorectal cancer (Fig. 4).

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, variations may be applied to the steps or order of steps of the methods and methods described herein without departing from the concept, spirit and scope of the present invention. It will be clear to those skilled in the art what is possible. More specifically, it will be apparent that certain agents, both chemically and physiologically related, may be substituted for those described herein while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Claims (45)

An isolated monoclonal antibody, said antibody specifically binding to 4-1BB,
(a) the first _VH CDR is identical to SEQ ID NO:3;
(b) the second _VH CDR is identical to SEQ ID NO:5;
(c) the third _VH CDR is identical to SEQ ID NO:7;
(d) the first V _L CDR is identical to SEQ ID NO: 11;
(e) the second V _L CDR is identical to SEQ ID NO: 13; and (f) the third V _L CDR is identical to SEQ ID NO: 15; or (a) the first V _H CDR is the sequence is identical to number 19,
(b) the second _VH CDR is identical to SEQ ID NO:21;
(c) the third _VH CDR is identical to SEQ ID NO:23;
(d) the first V _L CDR is identical to SEQ ID NO:27;
(e) the second V _L CDR is identical to SEQ ID NO: 29; and (f) the third V _L CDR is identical to SEQ ID NO: 31; or (a) the first V _H CDR is the sequence is identical to number 35,
(b) the second _VH CDR is identical to SEQ ID NO:37;
(c) the third _VH CDR is identical to SEQ ID NO:39;
(d) the first V _L CDR is identical to SEQ ID NO:43;
(e) the second V _L CDR is identical to SEQ ID NO: 45; and (f) the third V _L CDR is identical to SEQ ID NO: 47; or (a) the first V _H CDR is the sequence is identical to number 51;
(b) the second _VH CDR is identical to SEQ ID NO:53;
(c) the third _VH CDR is identical to SEQ ID NO:55;
(d) the first V _L CDR is identical to SEQ ID NO:59;
(e) the second V _L CDR is identical to SEQ ID NO:61; and (f) the third V _L CDR is identical to SEQ ID NO:63. the antibody
(a) the first _VH CDR is identical to SEQ ID NO:3;
(b) the second _VH CDR is identical to SEQ ID NO:5;
(c) the third _VH CDR is identical to SEQ ID NO:7;
(d) the first V _L CDR is identical to SEQ ID NO: 11;
(e) the second V _L CDR is identical to SEQ ID NO:13; and (f) the third V _L CDR is identical to SEQ ID NO:15. A claim comprising a _VH domain that is at least about 80% identical to the _VH domain of clone 54 (SEQ ID NO:2) and a _VL domain that is at least about 80% identical to the _VL domain of clone 54 (SEQ ID NO:10). 2. The antibody according to 2. A claim comprising a _VH domain that is at least about 90% identical to the _VH domain of clone 54 (SEQ ID NO:2) and a _VL domain that is at least about 90% identical to the _VL domain of clone 54 (SEQ ID NO:10). 2. The antibody according to 2. 3. The antibody of claim 2, comprising a _VH domain identical to the _VH domain of clone 54 (SEQ ID NO:2) and a _VL domain identical to the _VL domain of clone 54 (SEQ ID NO:10). the antibody
(a) the first _VH CDR is identical to SEQ ID NO: 19;
(b) the second _VH CDR is identical to SEQ ID NO:21;
(c) the third _VH CDR is identical to SEQ ID NO:23;
(d) the first V _L CDR is identical to SEQ ID NO:27;
(e) the second V _L CDR is identical to SEQ ID NO:29; and (f) the third V _L CDR is identical to SEQ ID NO:31. A claim comprising a _VH domain that is at least about 80% identical to the _VH domain of clone 135B (SEQ ID NO:18) and a _VL domain that is at least about 80% identical to the _VL domain of clone 135B (SEQ ID NO:26). 6. The antibody according to 6. A claim comprising a _VH domain that is at least about 90% identical to the _VH domain of clone 135B (SEQ ID NO:18) and a _VL domain that is at least about 90% identical to the _VL domain of clone 135B (SEQ ID NO:26). 6. The antibody according to 6. 7. The antibody of claim 6, comprising a _VH domain identical to the _VH domain of clone 135B (SEQ ID NO: 18) and a _VL domain identical to the _VL domain of clone 135B (SEQ ID NO: 26). the antibody
(a) the first _VH CDR is identical to SEQ ID NO:35;
(b) the second _VH CDR is identical to SEQ ID NO:37;
(c) the third _VH CDR is identical to SEQ ID NO:39;
(d) the first V _L CDR is identical to SEQ ID NO:43;
(e) the second V _L CDR is identical to SEQ ID NO:45; and (f) the third V _L CDR is identical to SEQ ID NO:47. A claim comprising a _VH domain that is at least about 80% identical to the _VH domain of clone 138 (SEQ ID NO:34) and a _VL domain that is at least about 80% identical to the _VL domain of clone 138 (SEQ ID NO:42). 11. The antibody according to 10. A claim comprising a _VH domain that is at least about 90% identical to the _VH domain of clone 138 (SEQ ID NO:34) and a _VL domain that is at least about 90% identical to the _VL domain of clone 138 (SEQ ID NO:42). 11. The antibody according to 10. 11. The antibody of claim 10, comprising a _VH domain identical to the _VH domain of clone 138 (SEQ ID NO:34) and a _VL domain identical to the _VL domain of clone 138 (SEQ ID NO:42). the antibody
(a) the first _VH CDR is identical to SEQ ID NO:51;
(b) the second _VH CDR is identical to SEQ ID NO:53;
(c) the third _VH CDR is identical to SEQ ID NO:55;
(d) the first V _L CDR is identical to SEQ ID NO:59;
(e) the second V _L CDR is identical to SEQ ID NO:61; and (f) the third V _L CDR is identical to SEQ ID NO:63. A claim comprising a _VH domain that is at least about 80% identical to the _VH domain of clone 49A (SEQ ID NO:50) and a _VL domain that is at least about 80% identical to the _VL domain of clone 49A (SEQ ID NO:58). 14. The antibody according to 14. A claim comprising a _VH domain that is at least about 90% identical to the _VH domain of clone 49A (SEQ ID NO:50) and a _VL domain that is at least about 90% identical to the _VL domain of clone 49A (SEQ ID NO:58). 14. The antibody according to 14. 15. The antibody of claim 14, comprising a _VH domain identical to the _VH domain of clone 49A (SEQ ID NO:50) and a _VL domain identical to the _VL domain of clone 49A (SEQ ID NO:58). 6. The antibody of any one of claims 1-5, wherein said antibody is recombinant. 2. The antibody of claim 1, wherein said antibody is IgG, IgM, IgA or an antigen-binding fragment thereof. 20. Any one of claims 1 to 19, wherein said antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv or single domain antibody. antibody. 2. The antibody of any one of claims 1, wherein the antibody is a human, humanized antibody or deimmunized antibody. 22. The antibody of any one of claims 1-21, wherein said antibody is conjugated to an imaging agent, chemotherapeutic agent, toxin or radionuclide. 23. A composition comprising the antibody of any one of claims 1-22 in a pharmaceutically acceptable carrier. 22. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the antibody of any one of claims 1-21. Clone 54 _VH domain CDRs 1-3 (SEQ ID NOS: 3, 5 and 7), Clone 135B _VH domain CDRs 1-3 (SEQ ID NOS: 19, 21 and 23), Clone 138 _VH domain CDRs 1-3 (SEQ ID NOS: 35, 37 and 39), or a recombinant polypeptide comprising an antibody _VH domain comprising CDRs 1-3 of the _VH domain of clone 49A (SEQ ID NOS: 51, 53 and 55). 54 _VL domain CDRs 1-3 (SEQ ID NOs: 11, 13 and 15), 135B _VL domain CDRs 1-3 (SEQ ID NOs: 27, 29 and 31), 138 _VL domain CDRs 1-3 (SEQ ID NOs: 43, 45 and 47), or a recombinant polypeptide comprising an antibody _VL domain comprising CDRs 1-3 of the _VL domain of 49A (SEQ ID NOS: 59, 61 and 63). Antibody _VH domain comprising CDR1-3 of _VH domain _of clone 54 (SEQ ID NOS:3, 5 and 7) and antibody VL domain comprising CDR1-3 of _VL domain of 54 (SEQ ID NO:11, 13 and 15) an antibody V _H domain comprising CDRs 1-3 of the V _H domain of clone 135B (SEQ ID NOs: 19, 21 and 23) and an antibody V _L comprising CDRs 1-3 of the V _L domain of 135B (SEQ ID NOs: 27, 29 and 31); domains; antibody _VH domain comprising CDR1-3 of the _VH domain of clone 138 (SEQ ID NOS:35, 37 and 39) and antibody V comprising CDR1-3 of the _VL domain of 138 (SEQ ID NOS:43, 45 and 47) or antibody _VH domain comprising CDR1-3 of the _VH domain of clone 49A (SEQ ID NOS:51, 53 and 55) and CDR1-3 of the _VL domain of 49A (SEQ ID _NOS :59, 61 and 63) A recombinant polypeptide comprising an antibody _VL domain. 28. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide of any one of claims 25-27. 28. A host cell comprising one or more polynucleotide molecules encoding the antibody of any one of claims 1-21 or the recombinant polypeptide of any one of claims 25-27. 30. The host cell of claim 29, wherein said host cell is a mammalian cell, yeast cell, bacterial cell, pili cell or insect cell. A method of producing an antibody, comprising:
(a) expressing in a cell one or more polynucleotide molecules encoding the _VL and _VH chains of the antibody of any one of claims 1-21;
(b) purifying said antibody from said cell. 22. A method of treating a subject with cancer, comprising administering to said subject an effective amount of the antibody of any one of claims 1-21. The cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, 33. The method of claim 32, which is colon cancer, rectal cancer or skin cancer. 33. The method of claim 32, wherein said antibody is in a pharmaceutically acceptable composition. 33. The method of claim 32, wherein said antibody is administered systemically. 33. The method of claim 32, wherein said antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. 33. The method of claim 32, further comprising administering at least a second anti-cancer therapy to said subject. 38. The method of claim 37, wherein said second anti-cancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. 38. The method of claim 37, wherein said second anti-cancer therapy comprises adoptive T cell therapy. 38. The method of claim 37, wherein said second anti-cancer therapy comprises immunotherapy. 41. The method of claim 40, wherein said immunotherapy is an immune checkpoint inhibitor. 42. The method of claim 41, wherein said at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-L1 antibody and/or an anti-PD1 antibody. 29. The method of claim 28, wherein the immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) binding antagonist, PDL1 binding antagonist or PDL2 binding antagonist. 44. The method of claim 43, wherein said PD-1 binding antagonist is a monoclonal antibody or antigen binding fragment thereof. Claim 43, wherein said PD-1 binding antagonist is nivolumab, pembrolizumab, CT-011 (pigilizumab), BMS-936559 (MDX-1105), MPDL328OA (atezolizumab) or AMP-224 (PD-L2 Fc fusion protein) The method described in .

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